LOCUS       sequence-482356-        8851 bp    DNA     circular SYN 07-AUG-2025
DEFINITION  Over-express Sc RFA1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8851)
  TITLE     O'Donnell lab plasmids 2025
REFERENCE   2  (bases 1 to 8851)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 7, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: Sc RFA[1+2]-pET
FEATURES             Location/Qualifiers
     source          1..8851
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             13..35
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    50..74
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(75..93)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     268..286
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(313..318,319..335,336..341)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     terminator      complement(516..563)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     RBS             2501..2523
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2538..2562
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(2563..2581)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2665..2684)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2890..2967
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     complement(2987..3006)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             5361..5552
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(5546..5568)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5654..5793
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5709..5728
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5808..5825)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5979..6567)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6059..6078)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6738..7529,7530..7598))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7361..7380
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      complement(7799..7846)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 tgccatggta tatctccttc ttaaagttaa acaaaattat ttctagaggg gaattgttat
       61 ccgctcacaa ttcccctata gtgagtcgta ttaatttcgc gggatcgaga tcattattga
      121 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
      181 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc
      241 attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtcttcaa
      301 gaattctcat gtttgacagc ttatcatcga taagctttaa tgcggtagtt tatcacagtt
      361 aaattgctaa cgcagtcagg caccgtgtat gaaatctaac aatgcgctca tcgtcatcct
      421 cggcaccgtc accctggatg ctgtaggcat aggcttggtt atgccggtac tgccgggcct
      481 cttgcgggat atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta
      541 gaggccccaa ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc
      601 tttcgggctt tgttagcagc cggatccatt aagctaacaa agccttggat aactcatcgg
      661 caagatagtc ggcttcagcc ctgtaattca agctgtgtag gttagcaacg gtatatctaa
      721 ttctgctttg atcattgtat gtatcctcac gcgctctaat cctaaagtca tattcgttca
      781 tttggatact ttgagtaatt tttgtgaatt cgttggggtc ttcttccttc aaagacatta
      841 atgtattagc atcaacaccc aataattgtt tagcttggtc gtcaaataaa gtgagccata
      901 gttgattggt ttcgtcaata attgatattg tcaagatgta tctccaattt ggccttgcat
      961 tattggtgtc gcacttctca catctccaag taccatcagg ctgttccaga actttcttat
     1021 tacaattctc attagaacag gcaggatatg caaaattatc aacttttaag aaacttatag
     1081 cagctttaac actaaaaaag tcacctttct cgcttcttcc tagattttca gcttgagctc
     1141 tagcaatagt aatacgctga gcaatgaatt ttgttaagct agcagccgat tgaccaccca
     1201 taccgggttc ttgctttaaa gtgatgaagt ttgcgttgcg gcccttggaa tcataccaac
     1261 cctttaaggc atatgcctca ggaatttctg gattcggaat cagggtacta gaaaatccca
     1321 tagacaaaga tttgccacca aaatccgtca cacgaacacc tttaatggca gcaacagaac
     1381 cttcaggaag gttgaaatca agggcttgct gattccatag gccaacagag atagaaaacc
     1441 cagagtcgtc aacaattgtg atgtcacgac gatcgaattt cttcccagcc cttgaagtta
     1501 gctcaaaatg tgggtttata gtttggataa taccgaggac gtctacgttg gaatttactt
     1561 cctggttctg aatagcatct agtttgatga aattgaaatg ggttttcgga acattacttt
     1621 catcgaaaca ttcttctata acagtgtctc tatccaaatt cagttcataa gggtgtgtta
     1681 gattagtaaa ttggggctta gctggttgga gttttgcctt tgatacatag tatactttgc
     1741 cttcttgtaa aatttcgtta aattttgtag caaaatcatt aaacgccgtg gctcggattt
     1801 ctccagaggt atccaagaag ttgacattga atagtttacc atcacctctt tgattgtgcc
     1861 acgttttaat ttctcccttg taggaaactc ttgctttgat agtccaaacg ttttggtatg
     1921 gagacagttg ttcgatggca aaaattggtc tggttttttg cgaattaggg ttttcattgg
     1981 cgaattttct ctcatttgca ttcaagtttg agtttgaatg cagcatatca gggacaccag
     2041 cattgctggc gtttgtttga ttggcaacat taccactgtc agttatatct tcgtctttta
     2101 aggtttcatt tggatgctct gagaaatagt tatccaaaaa agtactagtt tggttgacca
     2161 tatcagcacg cgactggacc aactcaaagt catctactaa aagaacgtat ttctttcttt
     2221 ccctgacaat agcaggttct gcaattatca cgcgaatgat atcacccctt tgtagttcca
     2281 ttgactggaa cttggatgca gcttggtttc tcaacagagc cttcatatgg taaataccat
     2341 cggaaatcat gatcaaattc tttctgttgc tgttagcccc atcagatttc ctggtgttat
     2401 aaacttgata aacgccaccg gtgggattat cgtacctttg cttattggtg aagatgctat
     2461 gaaaatcgcc cctcgaaagt tgaacactgg acatggtata tctccttctt aaagttaaac
     2521 aaaattattt ctagagggga attgttatcc gctcacaatt cccctatagt gagtcgtatt
     2581 aatttcgcgg gatcgagatc tcgatcctct acgccggacg catcgtggcc ggcatcaccg
     2641 gcgccacagg tgcggttgct ggcgcctata tcgccgacat caccgatggg gaagatcggg
     2701 ctcgccactt cgggctcatg agcgcttgtt tcggcgtggg tatggtggca ggccccgtgg
     2761 ccgggggact gttgggcgcc atctccttgc atgcaccatt ccttgcggcg gcggtgctca
     2821 acggcctcaa cctactactg ggctgcttcc taatgcagga gtcgcataag ggagagcgtc
     2881 gagatcccgg acaccatcga atggcgcaaa acctttcgcg gtatggcatg atagcgcccg
     2941 gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag
     3001 tatgccggtg tctcttatca gaccgtttcc cgcgtggtga accaggccag ccacgtttct
     3061 gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc tgaattacat tcccaaccgc
     3121 gtggcacaac aactggcggg caaacagtcg ttgctgattg gcgttgccac ctccagtctg
     3181 gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt
     3241 gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg
     3301 cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta actatccgct ggatgaccag
     3361 gatgccattg ctgtggaagc tgcctgcact aatgttccgg cgttatttct tgatgtctct
     3421 gaccagacac ccatcaacag tattattttc tcccatgaag acggtacgcg actgggcgtg
     3481 gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc attaagttct
     3541 gtctcggcgc gtctgcgtct ggctggctgg cataaatatc tcactcgcaa tcaaattcag
     3601 ccgatagcgg aacgggaagg cgactggagt gccatgtccg gttttcaaca aaccatgcaa
     3661 atgctgaatg agggcatcgt tcccactgcg atgctggttg ccaacgatca gatggcgctg
     3721 ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg
     3781 ggatacgacg ataccgaaga cagctcatgt tatatcccgc cgttaaccac catcaaacag
     3841 gattttcgcc tgctggggca aaccagcgtg gaccgcttgc tgcaactctc tcagggccag
     3901 gcggtgaagg gcaatcagct gttgcccgtc tcactggtga aaagaaaaac caccctggcg
     3961 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
     4021 cagtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtaag ttagctcact
     4081 cattaggcac cgggatctcg accgatgccc ttgagagcct tcaacccagt cagctccttc
     4141 cggtgggcgc ggggcatgac tatcgtcgcc gcacttatga ctgtcttctt tatcatgcaa
     4201 ctcgtaggac aggtgccggc agcgctctgg gtcattttcg gcgaggaccg ctttcgctgg
     4261 agcgcgacga tgatcggcct gtcgcttgcg gtattcggaa tcttgcacgc cctcgctcaa
     4321 gccttcgtca ctggtcccgc caccaaacgt ttcggcgaga agcaggccat tatcgccggc
     4381 atggcggccg acgcgctggg ctacgtcttg ctggcgttcg cgacgcgagg ctggatggcc
     4441 ttccccatta tgattcttct cgcttccggc ggcatcggga tgcccgcgtt gcaggccatg
     4501 ctgtccaggc aggtagatga cgaccatcag ggacagcttc aaggatcgct cgcggctctt
     4561 accagcctaa cttcgatcat tggaccgctg atcgtcacgg cgatttatgc cgcctcggcg
     4621 agcacatgga acgggttggc atggattgta ggcgccgccc tataccttgt ctgcctcccc
     4681 gcgttgcgtc gcggtgcatg gagccgggcc acctcgacct gaatggaagc cggcggcacc
     4741 tcgctaacgg attcaccact ccaagaattg gagccaatca attcttgcgg agaactgtga
     4801 atgcgcaaac caacccttgg cagaacatat ccatcgcgtc cgccatctcc agcagccgca
     4861 cgcggcgcat ctcgggcagc gttgggtcct ggccacgggt gcgcatgatc gtgctcctgt
     4921 cgttgaggac ccggctaggc tggcggggtt gccttactgg ttagcagaat gaatcaccga
     4981 tacgcgagcg aacgtgaagc gactgctgct gcaaaacgtc tgcgacctga gcaacaacat
     5041 gaatggtctt cggtttccgt gtttcgtaaa gtctggaaac gcggaagtca gcgccctgca
     5101 ccattatgtt ccggatctgc atcgcaggat gctgctggct accctgtgga acacctacat
     5161 ctgtattaac gaagcgctgg cattgaccct gagtgatttt tctctggtcc cgccgcatcc
     5221 ataccgccag ttgtttaccc tcacaacgtt ccagtaaccg ggcatgttca tcatcagtaa
     5281 cccgtatcgt gagcatcctc tctcgtttca tcggtatcat tacccccatg aacagaaatc
     5341 ccccttacac ggaggcatca gtgaccaaac aggaaaaaac cgcccttaac atggcccgct
     5401 ttatcagaag ccagacatta acgcttctgg agaaactcaa cgagctggac gcggatgaac
     5461 aggcagacat ctgtgaatcg cttcacgacc acgctgatga gctttaccgc agctgcctcg
     5521 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag
     5581 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg
     5641 gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg tatactggct
     5701 taactatgcg gcatcagagc agattgtact gagagtgcac cattgcggtg tgaaataccg
     5761 cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc gctcactgac
     5821 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     5881 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     5941 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     6001 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     6061 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     6121 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     6181 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     6241 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     6301 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     6361 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
     6421 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     6481 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     6541 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     6601 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     6661 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     6721 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     6781 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     6841 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     6901 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     6961 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     7021 gttaatagtt tgcgcaacgt tgttgccatt gctgcaggca tcgtggtgtc acgctcgtcg
     7081 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     7141 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     7201 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     7261 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     7321 atgcggcgac cgagttgctc ttgcccggcg tcaacacggg ataataccgc gccacatagc
     7381 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     7441 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     7501 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     7561 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatagctt
     7621 taatgcggta gtttatcaca gttaaattgc taacgcagtc aggcaccgtg tatgaaatct
     7681 aacaatgcgc tcatcgtcat cctcggcacc gtcaccctgg atgctgtagg cataggcttg
     7741 gttatgccgg tactgccggg cctcttgcgg gatatccgga tatagttcct cctttcagca
     7801 aaaaacccct caagacccgt ttagaggccc caaggggtta tgctagttat tgctcagcgg
     7861 tggcagcagc caactcagct tcctttcggg ctttgttagc agccggatcc atgcatctta
     7921 ccccttcttg gaacgggttc cattttccac tctatttccg gcaaattttg gtacttacct
     7981 agtagaaata cacaaacgaa tactaagaaa tgctaaaaaa ataatctata tattttcata
     8041 gggcaaagaa gttattgtca tcaaaagttg ggtagataaa accctggtca gtcaaggtcg
     8101 tacagcagtt tctgacggta gtttcatcca aattcaagga ttgcgagatc aatggaatgg
     8161 gaacagcgaa tgaattagcg tctttgccct cacattgctt cttacaaaat tctagaattc
     8221 tttgtaacgg actggagccg gaagatgtat cgttatcatc cttgacaaat aatgattgcc
     8281 cattgttgtt ggatgcactc tccaaaggtt gtttcatcat tccactggct atggaatgac
     8341 atttgatgac ttccaagtga tgcgtcaaca cttcattgaa tgaatctatg ggcttaatca
     8401 ccgcatactg tatatttttc ttaccaccaa actctttcaa agcaccaaaa acttttacgt
     8461 aaccgccaat ttcaaattgt tgggcgactt gcgaaccata acctttacca gaagagtcat
     8521 cgttaccggc agccaagtca tttgcatctt cgctccattt tctcacttct atttgaccag
     8581 ttccatcctc aatagttaaa aaaatatttg cagtatggtc tgtaatgttt ctcaccacac
     8641 ctacaaaaca aacgtgatga agttcttggt tatgcgaaac gaaggggccg tcctgaatat
     8701 cctgtttgga ctctagaatt tgtttgatcg tcacaggtgt caaggtgtta actctagtgt
     8761 tagtttccga ctccccacta cctgggcggg actcagagtt ctcaaagcca ccgcccgtta
     8821 ctgatgaata ttcgttatat ggttgatagg t
//
